Direct reprogramming of human epithelial cells into ... - bioRxiv

bioRxiv preprint first posted online May. 4, 2018; doi: http://dx.doi.org/10.1101/314658. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.

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Direct reprogramming of human epithelial cells into organoids by miR-106a-3p

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Delom F.1*, Puceat M.2, and Fessart D.1*

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1 Univ. Bordeaux, F-33000 Bordeaux, France.

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2 Université Aix-Marseille, INSERM UMR_1251, F-13385 Marseille, France.

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* Corresponding author:

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Delphine Fessart - Bordeaux University - 146, Léo-Saignat – 33076 Bordeaux – France. Phone:

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(00 33) +5 56 33 04 31. Fax: (00 33) +5 56 33 33 30. E-mail: [email protected]

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Frederic Delom - Bordeaux University - 146, Léo-Saignat – 33076 Bordeaux – France. Phone:

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(00 33) +5 56330431. Fax: (00 33) +5 56333330. E-mail: [email protected]

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Lead contact: Delphine Fessart, [email protected]

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Abstract

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Organoids development relies on the self-organizing properties of adult stem cells to create structures

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which recapitulate the architecture, functionality, and genetic signature observed in original tissues.

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Little is known about of the exact nature of the intrinsic cell properties at the origin of organoid

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generation, and of the signaling pathways governing their differentiation. Herein, we carried out a

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functional microRNA screen to identify miRNAs at the origin of organoid generation from human

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epithelial cell culture. We uncover miR-106a-3p that initiates and promotes organoids. This miRNA

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acts as a master inducer of the expression of the three core pluripotency transcription factors

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(NANOG, OCT4 and SOX2) through the regulation of a set of 10 genes, and thus strengthening the

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reprogramming and cell differentiation of human epithelial cells into organoids. These data

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demonstrate that organoids can be directly generated from human epithelial cells by only one miRNA:

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miR-106a-3p. Hence, we appear to have identified a new determinant of organoid identity, which

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plays a role in reprogramming, cell differentiation and tissue engineering.

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Introduction

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Three-dimensional (3D) human organoid culture models are appealing tools to study

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pathophysiological processes. These models have been described, by us and others, for the

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lung

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like, reflecting the ability of organoid culture conditions to prompt cells to self-organize into

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structures mimicking the architecture of the organ from which they were derived. In contrast

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to organ explants, organoids can arise from a single primary cell

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as well as for numerous other tissues

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. The term "organoid" literally means organ-

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, thereby allowing the

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generation of human organoids from biopsies . Non-tumor organoids are thought to arise

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from adult stem cells (aSCs), and therefore should in theory be capable of self-renewal and

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differentiation. Consistent with this idea, primary cells enriched with known progenitor/stem

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cell markers are more efficient at forming organoids than the general cell population

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However, currently there is a lack of understanding of the underlying epigenetic and genetic

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mechanisms that control organoid-initiating frequency, self-renewal and differentiation during

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organogenesis.

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.

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To better understand mammalian development, as well as to exploit the tremendous

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therapeutic potential of organoid models, it is necessary to identify and characterize the

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genetic mechanisms governing the fate of aSCs. MicroRNAs (miRNA) have been recently

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shown to play an important role in regulating stem cell self-renewal and differentiation 15. In

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general, one gene can be repressed by multiple miRNAs and one miRNA may repress

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multiple target genes, which results in the formation of complex regulatory networks. In a

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wide variety of developmental processes, miRNAs finely tune or restrict cellular identities by

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targeting important transcription factors or key pathways

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the contribution of miRNA-mediated gene regulation in the enrichment of progenitor/stem

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cell markers. This would allow us to better characterize the mechanisms responsible for

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controlling the initiating cell sub-population and thus improving tissue-specific organoid

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growth conditions. Consequently, we performed a miRNA screen in human primary epithelial

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cells to identify the mediators influencing the initiation of stem cell derived organoids. We

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identified a previously uncharacterized miRNA, miR-106a-3p, and its target genes that play a

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key role in such process. Using a gain of function approach, we discovered that the

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endogenous levels of three core transcription factors (OCT4, SOX2 and NANOG) were post-

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transcriptionally controlled by miR-106a-3p in human aSCs. Moreover, we discovered that

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miR-106a-3p is necessary and sufficient to fine tune the differentiation process, and thus the

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pluripotent state through a specific transcriptional regulatory network. Overall, our results

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. Hence, we sought to investigate

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highlight the importance of miR-106a-3p in the initiation of stem-cell derived organoids and

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provide some clues about the mechanism underlying organogenesis.

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Results

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Organoid culture of Human Epithelial Cells exhibits a CD44high/CD24low phenotype

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This study was initiated to identify organoid-initiating epithelial cell subpopulations 13

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that specify stem/progenitor cell functions in epithelial cells

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characteristics of stem cell is to be rare immortal cells within a mass culture that can both self-

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renew by dividing and give rise to many cell types. First, we characterized the properties of

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human primary mammary epithelial cells (HMEC) grown in 3D compared to conventional 2D

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culture. As a control, cells were grown under organoid culture conditions as we previously

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described

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Approximately 3% of cells present in the culture featured the capacity to reconstitute an

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organoid (Figure 1A), suggesting the presence of stem cells within the mass culture. Next, the

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self-renewal capacity of organoid-initiating cells was assessed by serial organoid formation

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from passage 5 to passage 11 (Figure 1B). Cells progressively lost self-renewal ability to form

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organoids upon serial propagation (Figure 1B), consistent with previously described loss of

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self-renewal potential of primary epithelial stem cells after few passages 14.

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. One of the main

and the cell lines tested formed 3D-structured human organoids (Figure 1A).

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Previous studies have reported that human mammary epithelial cells with

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CD44high/CD24low phenotype have the highest progenitor ability compared to all other

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stem/progenitor subpopulations 7. Therefore, we analyzed the expression of CD44 and CD24

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in 2D cell culture compared to 3D using flow cytometry (Figure 1C). In 2D cell culture, 85%

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expressed both CD24 and CD44 at high levels and 14% expressed CD24 at low levels

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together with high levels of CD44 (Figure 1C, Top panel). In contrast, 3D cell culture showed

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more than 3-fold increase in CD44high/CD24low phenotype cells (~49%) compared to 2D

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(~14%) (Figure 1C; lower panel, p=0.0268, n=3). We then analyzed the cells from 2D culture

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using standard immunofluorescence (Figure 1D) to determine the expression of CD24 and

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CD44 cell-surface markers. The overlaid images showed a mix of cell populations: CD24

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cells (green), CD44 cells (red) and CD44/CD24 co-expressing cells (yellow) (Figure 1D).

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Together, these results indicate that cells grown as organoids acquired a CD44high/CD24low

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expression pattern similar to stem/progenitor cell that can be used for further screening.

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A miRNA screening approach to selectively favor organoids formation

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To investigate whether miRNA-mediated gene regulation could promote organoid

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formation, we monitored, as a tool, the expression of CD44 and CD24 following miRNA

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transfection into HMEC cells (Figure 2A-D). Following quantitative image analysis of

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>100,000 cells at Passage 6 (P6), frequency distributions of CD44 intensity were compared in

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mass culture (whole population), and mass culture exposed to CD44 siRNA (Figure 2A) or

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exposed to CD24 siRNA (Figure 2B). CD44 and CD24 levels were lower in siRNA-depleted

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cells in comparison to the whole population, which validates the specificity of our assay

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(Figure 2A-B). To identify miRNAs that play a role in the enrichment of CD44high/CD24low

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cell phenotype, we performed an unbiased functional screen for miRNAs that modulate

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CD44/CD24 phenotypes in HMEC (Figure 2C). Using an approach similar to our genome-

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wide small interfering RNA (siRNA) screen for p16 modulators 2, we transfected actively

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proliferating cells (Passage 6, P6) with 837 miRNAs. siRNA targeting siGLO (‘cyclophilin

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B’; PPIB), CD44 or CD24 served as controls. We assigned cut-off values to define miRNA

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hits based on CD44 and CD24 cell density. The raw screening data and quantitation of each

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phenotypic criterion are shown in Figure 2D. This strategy revealed that the miR-106a-3p

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shifts primary cells into a CD44high/CD24low phenotype. This miRNA is a paralogue of the

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miR-17/92 cluster (Figure 2E). Next, to further confirm the results, we performed a secondary

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screen of the whole family cluster (Figure 2E). Twenty-eight miRNAs belonging to the

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cluster were retested, in triplicate, using the same method as in the primary screen (Figure

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1C). A total of 4 hits were scored as those miRNAs with Z-factor >2 (Figure 2F) and

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prompted a shift in the CD44high/CD24low population (Figure S1B). The top hit was miR-

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106a-3p (Figure 2F and Figure S1A). We then confirmed miR-106a-3p induced a

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CD44high/CD24low phenotype using flow cytometry based on the expression of CD44 and

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CD24 (Figure S1B and Figure 2G). In cells expressing the control mimic, the

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CD44high/CD24low population (CD24-/CD44+) was ~10% of the total cell population

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(Figure 2G). Conversely, we observed a 5-fold increase of CD44high/CD24low population,

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~50% of the total cell population, in cells transfected with a mimic miR106a-3p (Figure 2G).

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In parallel, to correlate these data with organoid development, we assessed the

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organoid-initiating frequency of each of the 28 miRNAs (miR-17/92 cluster) (Figure 2E). Out

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of the total of 7 positive hits (Figure 2H), miR-106a-3p displayed the highest organoid-

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initiating frequency (Figure 2H). Taken together, these results show that miR-106a-3p (Figure

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2I), is the only miRNA that exhibits the two properties of 1) enriching CD44high/CD24low

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cells and 2) favoring organoid development. 6


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The development of human organoids is driven by miR-106a-3p.

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Next, we questioned whether miR-106a-3p is endogenously expressed in organoids

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compared to 2D culture. We found that only the 3D culture of organoids expressed miR-106a-

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3p, thus reinforcing its potential role in organoid formation (Figure 3A). To further study

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miR-106a-3p function, we generated retroviral vectors of miR-106a as previously described 24

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evaluated its stable expression in HMECs (Figure 3B-E). First, we examined the expression of

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miR-106a-5p and miR-106a-3p using RT-qPCR in control (miR-Vector) and miR-106a-

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infected cells (Figure 3B) and observed that miR-106a-5p was expressed in both conditions.

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On the contrary, both RT-qPCR and in situ hybridization showed that miR-106a-3p was

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exclusively expressed in miR-106a cells (Figure 3B-C) at levels similar to those observed in

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3D cultures (Figure 3A).

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As expected, miR-106a stable overexpression greatly increased organoid-initiating

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frequency (Figure 3D). To further evaluate the impact of miR-106a on organoid architecture,

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organoids were analyzed using confocal microscopy. Apoptotic cells are present in organoids

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during lumen development

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Caspase-3, demonstrated that miR-106a did not impact on luminal apoptosis during

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organogenesis (Figure 3E, Caspase-3). Moreover, organoids are characterized by a well-

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defined cell/Matrigel interface with a myoepithelial layer, which was not impacted by miR-

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106a overexpression (Figure 3E, CD44 and p63). In addition, organoids expressed β-catenin

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(cell junction marker) adjacent to the plasma membranes and miR-106a overexpression did

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not show any effect on its localization and did not disrupt cell junctions (Figure 3E, β-

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catenin). These results demonstrate that miR-106a does not disrupt the structure of organoids.

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. Immunofluorescence staining for the apoptosis marker,

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To test miR-106a-3p and miR-106a-5p individual functions on capacity of organoid-

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initiating cells, miR-106a-3p or miR-106a-5p mimics were transfected in HMEC cells (Figure

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3F-G). We examined miR-106a-5p and miR-106a-3p expression by RT-qPCR and observed

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that miR-106a-5p is expressed both in control, miR-106a-5p and miR-106a-3p cells (Figure

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3F). As expected, miR-106a-3p is only expressed in miR-106a-3p transfected cells (Figure

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3F). Next, we studied the individual role of miR-106a-5p and miR-106a-3p overexpression on

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organoid-initiating frequency (Figure 3G). The overexpression of miR-106a-3p significantly

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increases organoids number by about 5-fold compared to control and miR-106a-5p (Figure

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3G). Our results indicate that miR-106a-5p does not impact on organoids frequency,

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demonstrating the specific requirement of miR-106a-3p to mediate the self-renewal capacity 7


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of organoid-initiating cells (Figure 3G). Taken together, these results demonstrate that i) miR-

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106a-3p expression's is only restricted to organoids, and ii) miR-106a-3p, but not miR-106a-

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5p, is required for the development of human organoids.

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Identification of miR-106a-3p targets

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Since a single miRNA can potentially target hundreds of genes

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, we next cross-

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referenced the predicted targets of miR-106a-3p using four different algorithms (miRanda,

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miRDB, DianaMT and miRWalk). We found 67 genes common to the four algorithms (Figure

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4A). In parallel, to study the effect of the over-expression of miR-106a-3p on global gene

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expression patterns, we isolated total RNA from HMEC cells transfected with miR-106a-3p

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mimic and performed microarray analysis (HG-U133 Plus 2.0). The results indicated that

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transfection of miR-106a-3p induced significant changes in the expression of 6465 genes (p

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value